Expression of Myosin Light Chain Kinase and Phosphorylation of Myosin-II Light Chain (MLC) in Cultured Bovine Trabecular Meshwork (TM) Cells

Abstract

Abstract: : Purpose: Several pharmacological agents, which affect contractility of TM strips mounted in vitro, alter resistance to aqueous humor outflow across the TM in vivo. The molecular components underlying the contractility of TM cells are not well understood. This study has examined the activity of Rho kinase in the regulation of Myosin II regulatory light chain phosphorylation in cultured bovine TM cells. Methods: Expression of Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) isoforms (smooth muscles and non-muscle forms), a key component in the direct pathway leading to phosphorylation of MLC, was identified by Western blotting. MLC phosphorylation was induced by exposure of TM cells to thrombin (1 unit/ml), well known to elicit MLC phoshorylation in a variety of cell types. Phosphorylated and unphosphorylated MLC were separated by urea-glycerol gel and identified by Western blotting. MLCK was inhibited by ML-7 (50 microM). Rho kinase, an inhibitor of myosin phosphatase, was inhibited by Y-27632. Altered contractility in response to Y-27632 was assessed through isometric tension measurements of bovine TM strips perfused in vitro. Results: Western blot showed expression of both 130 kd- and 220 kd- isoforms of MLCK corresponding to isoforms found in smooth muscle and non-muscle cells, respectively. RT-PCR showed a positive band indicated expression of MLCK in bovine TM. MLC20 phosphorylation was induced by both NaF and thrombin. Thrombin-induced phosphorylation was inhibited by Rho kinase inhibitor Y-27632 (100 microM for 30 min) significantly. The effect of ML-7 (50 microM for 30 min), MLCK inhibitor, was not significant. Bovine TM strips pre-contracted by exposure to carbachol (set to 100%) showed dose dependent relaxation upon exposure to Y-27632 (from 100% carbachol: 7.2 +/- 2.9% at 1 microM; n = 9 and 25.6 +/- 5.5% at 5 microM; n = 7). Conclusions: (1) Thrombin-induced phosphorylation of MLC20 in TM cells is mediated by Rho kinase. (2) Y-27632 induced relaxation of TM strips is consistent with the prevention of myosin phosphatase activity by Rho kinase. (3) Reported Y-27632-induced reduction in intraocular pressure, therefore, is through relaxation of TM.

Keywords: trabecular meshwork • pharmacology • outflow: trabecular meshwork